Development and validation of a rapid and sensitive LC-ESI-MS/MS method for ondansetron quantification in human plasma and its application in comparative bioavailability study.

The validation of a high throughput and specific method using a high-performance liquid chromatography coupled to electrospray (ES+) ionization tandem triple quadrupole mass spectrometric (LC-ESI-MS/MS) method for ondansetron quantification in human plasma is described. Human plasma samples were extracted by liquid-liquid extraction (LLE) using methyl tert-butyl ether and analyzed by LC-ESI-MS/MS. The limit of quantification was 0.2 ng/mL and the method was linear in the range 0.2-60 ng/mL. The intra-assay precisions ranged from 1.6 to 7.7%, while inter-assay precisions ranged from 2.1 to 5.1%. The intra-assay accuracies ranged from 97.5 to 108.2%, and the inter-assay accuracies ranged from 97.3 to 107.0%. The analytical method was applied to evaluate the relative bioavailability of two pharmaceutical formulations containing 8 mg of ondansetron each in 25 healthy volunteers using a randomized, two-period crossover design. The geometric mean and respective 90% confidence interval (CI) of ondansetron test/reference percent ratios were 90.15% (81.74-99.44%) for C(max) and 93.11% (83.01-104.43%) for AUC(0-t). Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) and AUC(0-inf), it was concluded that the test formulation is bioequivalent to the reference one with respect to the rate and extent of absorption of ondansetron.

Simultaneous determination of losartan and hydrochlorothiazide in human plasma by LC/MS/MS with electrospray ionization and its application to pharmacokinetics.

A method based on a simple liquid-liquid extraction (LLE) followed by high-performance liquid chromatography with negative ion electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) detection was developed for the simultaneous determination of losartan (LOS) and hydrochlorothiazide (HCTZ) in human plasma, using valsartan (VAL) and chlorthalidone (CHTD) as an internal standard, respectively. The acquisition was performed in multiple reactions monitoring (MRM) and the limit of quantification was 4 ng/mL for both LOS and HCTZ. The method was linear in the studied range (4-800 ng/mL for LOS and 4-500 ng/mL for HCTZ). The intra-assay precisions ranged from 2.6-11.9% for LOS and 1.4-8.2% for HCTZ, while the inter-assay precisions ranged from 1.0-8.0% for LOS and 2.5-7.7% for HCTZ. The intra-assay accuracies ranged from 91.3 to 107.6% for LOS and 91.5 to 105.8% for HCTZ, while the inter-assay accuracies ranged from 99.9 to 106.4% for LOS and 97.4 to 101.4% for HCTZ. The analytical method was applied to a bioequivalence study, in which 28 healthy adult volunteers (14 men) received single oral doses (100 mg LOS + 25 mg HCTZ) of reference and test formulations, in an open, two-period, balanced randomized, crossover protocol. Based on the 90% confidence interval of the individual ratios for Cmax and AUC0-inf, it was concluded that the test formulation is bioequivalent to the reference Hyzaar formulation with respect to the rate and extent of absorption of both LOS and HCTZ.

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study.

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.

A rapid and sensitive method for simultaneous determination of lamivudine and zidovudine in human serum by on-line solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry detection.

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the simultaneous determination of lamivudine (3TC) and zidovudine (AZT) in human serum, using didanosine (ddI) as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 230.0 --> 111.8 for 3TC, m/z 268.1 --> 126.8 for AZT, and m/z 237.2 --> 136.8 for ddI. The limits of detection and quantitation were 3 and 10 ng/mL for 3TC, and 5 and 15 ng/mL for AZT. The method was linear in the studied ranges (10-1500 ng/mL for 3TC and 15-3000 ng/mL for AZT), with r(2) > 0.99 for each drug, and the run time was 4 min. The intra-assay precisions (%) were in the ranges 1.9-8.7 (3TC) and 2.2-8.9 (AZT), the inter-assay precisions were in the ranges 2.6-9.0 (3TC) and 4.2-8.1 (AZT), and the intra- and inter-assay accuracies were >97% for both drugs. The absolute recoveries were 95-99% for 3TC (45, 600 and 1200 ng/mL) and 104-112% for AZT (45, 1000 and 2400 ng/mL). The analytical method was applied to a bioequivalence study in which 24 healthy adult volunteers received single oral doses of the reference formulation and two test combined AZT/3TC tablets, in an open, three-period, balanced, randomized, crossover protocol. Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (extrapolated area under the serum concentration vs. time curve from time zero to infinity), it was concluded that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of absorption of both 3TC and AZT.

Prednisolone: limited sampling strategies for estimating pharmacokinetic parameters.

To develop limited-sampling strategy (LSS) models for estimating prednisolone's area under plasma concentration versus time curve (AUC(0-infinity)), its maximum concentration in plasma (C(max)), and total clearance (CL/F). Healthy subjects (n = 24), enrolled in a bioequivalence study, received 20 mg PO of the prodrug prednisone as reference and test tablets, and plasma prednisolone concentrations (n = 576) were measured by a validated HPLC assay. A linear regression analysis of AUC(0-infinity), C(max), CL/F, and log(CL/F) against the plasma prednisolone concentrations for the reference formulation was carried out to develop LSS models to estimate these parameters. The LSS models were validated on the test formulation data sets and on simulated sets generated by the software ADAPT II. LSS models based on a single [1.5 hours for C(max) and 7 hours for AUC(0-infinity), CL/F, and log(CL/F)] plasma sample, accurately estimated (R2 = 0.84-0.97, mean bias < 1%; mean precision < 10%) these pharmacokinetic parameters. Validation tests indicated that the most informative single-point LSS models developed for the reference formulation provide precise estimates (R(2) > 0.83; mean bias < 3%; mean precision < 10%) of the corresponding pharmacokinetic parameters for the test formulation. LSS models based on the two most informative sampling points (1.5 and 7 hours) were required for accurate estimates (R(2) > 0.87; mean bias < 6%; mean precision < 8%) of prednisolone's C(max), AUC(0-infinity), CL/F, and log(CL/F) for the simulated data sets. Finally, bioequivalence assessment of the prednisone formulations, based on LSS-derived AUC(0-infinity) and C(max) values provided results identical to those obtained using the original values for these parameters. One- and 2-point LSS models provided accurate estimates of prednisolone's C(max), AUC(0-infinity), and CL/F, following single oral doses of prednisone, and allowed correct assessment of bioequivalence between two prednisone formulations.

Determination of stavudine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometry: application to a bioequivalence study.

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of stavudine in human serum, using didanosine as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode. The method was linear over the studied range (10-2000 ng/mL), with r(2) > 0.99, and the run time was 4 min. The intra- and inter-assay precisions (%) were in the ranges 0.1-13.6 and 2.6-9.9, respectively, and the intra- and inter-assay accuracies were >92%. The absolute recoveries were approximately 100% (10 ng/mL), 98% (30 ng/mL), 105% (750 ng/mL) and 105% (1500 ng/mL). The limits of detection and quantitation were 4 and 10 ng/mL, respectively. The analytical method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (40 mg) of reference and two test stavudine formulations, in an open, three-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration), AUC(0-10) and AUC(0-inf) (areas under the serum concentration vs. time curve from time zero to 10 h and to infinity, respectively), were in the range 80-125%, which supports the conclusion that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of stavudine absorption.

Determination of didanosine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study.

A method based on solid-phase extraction coupled to liquid chromatography with positive ion electrospray ionization and tandem mass spectrometric detection was developed for the determination of didanosine in human serum, using lamivudine as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions m/z 237 --> 136.7 for didanosine and m/z 230 --> 111.7 for lamivudine. The method was linear over the range studied (10-1500 ng ml(-1)), with r(2) > 0.98, and the run time was 5 min. The intra- and inter-assay precisions were < or =10% and the intra- and inter-assay accuracies were >95%. The absolute recoveries were 99.8% (10 ng ml(-1)), 98.4% (30 ng ml(-1)), 91.5% (700 ng ml(-1)) and 94.7% (1200 ng ml(-1)). The limits of detection and quantitation were 5 and 10 ng ml(-1), respectively. The method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (200 mg) of reference and test didanosine formulations (buffered powder for oral solutions), in an open, two-way, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (area under the serum concentration versus time curve from time zero to infinity) were within the range 80-125%, which supports the conclusion that the two formulations are bioequivalent regarding the rate and extent of didanosine absorption.

Combined chromatographic methodology for antidoping control in horse urine

A combination of chromatographic techniques is described for the screening of drugs in horse urine for antidoping control purposes. The procedure consists of liquid-liquid base extraction with analysis by HPTLC and GC-NPD and liquid-liquid acidic extraction followed by HPTLC and HPLC. The results of 86 drugs analyzed by these procedures, as well as limits of detection of the drugs in each technique are presented, The use of ELISA tests are recommended for the screening of drugs which would not be detected by the chromatographic proposed procedure. The results show that the proposed combination of techniques enables an efficient antidoping control of racing horses.

Determinaçäo de hidrocortisona em urina de cavalo de corrida empregando cromatografia a líquido de alta eficiência com detector de arranjo de diodos / Hydrocortisone determination in horseracing urine using high-performance liquid chromatography with diode arrangememt detection

A presença de hidrocortisona (cortisol) em amostras de urina de cavalos submetidas à análise de controle antidopagem pode ser decorrência não apenas da secreção endógena dessa substância pelo cortéx da adrenal, mas também da administração deste fármaco. Devido às suas propriedades antiinflamatórias, a hidrocortisona é enquadrada no Grupo II de substâncias proibidas pelo Código Nacional de Corridas. Este trabalho apresenta os resultados obtidos para um método de confirmação da presença de hidrocortisona em urina eqüina, o qual utiliza extração liquído-liquído, seguida de purificação por CCD e determinação por HPLC. Os resultados mostraram que o método empregado é adequado ao seu propósito.

Validacao de metodos cromatograficos em analises toxicologicas / Validation of chromatographic methods in toxicological analyses

A identificacao e quantificacao de xenobioticos em amostras biologicas pressupoe a utilizacao de metodologia cuja validacao tenha sido devidamente estabelecida. Agencias internacionais, tais como, Environmental Protection Agency, Food and Drug Administration, American Industry Hygiene Association, etc., responsaveis por programas de seguranca da qualidade em analises toxicologicas exigem relatos dos processos de validacao. A maxima "faca o que esta escrito e escreva o que e feito" mostra a enfase dada a tal documentacao. A validacao de um metodo analitico inclui todos os procedimentos realizados para garantir a qualidade dos dados produzidos. Independentemente do processo analitico a ser utilizado, preconiza-se um protocolo composto, basicamente, de tres estagios. O primeiro relaciona-se a procedimentos de afericao de instrumentos e calibracao de equipamentos visando assegurar a confiabilidade das medidas. O segundo diz respeito ao estudo da estabilidade do xenobiotico na matriz, considerando as condicoes de armazenamento e ciclos de congelamento e descongelamento, recuperacao, precisao, exatidao, limites de deteccao e quantificacao, especificidade e intervalo dinamico. O terceiro estagio baseia-se nos procedimentos adotados pelo laboratorio para garantir que o procedimento, como um todo, esta sob controle. Neste trabalho descrevem-se principios praticos e recomendacoes gerais no sentido de indicar diretrizes para o estabelecimento de protocolos que atestem a validade do resultado analitico